Synthesis, anticancer evaluation, molecular docking and ADME study of novel pyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]pyrimidines as potential tropomyosin receptor kinase A (TrKA) inhibitors

The starting compound 3-amino-1,7-dihydro-4H-pyrazolo[4,3-c]pyridine-4,6(5H)-dione (1) is reacted with each of diketone and β-ketoester, forming pyridopyrazolo[1,5-a]pyrimidines 4a,b and 14a,b, respectively. The compounds 4 and 14 reacted with each of aromatic aldehyde and arenediazonium salt to give the respective arylidenes and arylhydrazo derivatives, respectively. The structure of the new products was established using spectroscopic techniques. The cytotoxic activity of selected targets was tested in vitro against three cancer cell lines MCF7, HepG2 and HCT116. The data obtained from enzymatic assays of TrKA indicated that compounds 7b and 16c have the strongest inhibitory effects on TrKA with IC50 = 0.064 ± 0.0037 μg/ml and IC50 = 0.047 ± 0.0027 μg/ml, respectively, compared to the standard drug Larotrectinib with IC50 = 0.034 ± 0.0021 μg/ml for the HepG2 cancer cell line. In cell cycle analysis, compounds 7b, 15b, 16a and 16c caused the greatest arrest in cell cycle at the G2/M phase. In addition, compound 15b has a higher apoptosis-inducing effect (36.72%) than compounds 7b (34.70%), 16a (21.14) and 16c (26.54%). Compounds 7b, 16a and 16c were shown fit tightly into the active site of the TrKA kinase crystal structure (PDB: 5H3Q). Also, ADME study was performed on some selected potent anticancer compounds described in this study. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-024-01166-7.


Introduction
Cancer is the growth of cells in certain parts of the body that grow out of control and can invade other tissues.Cancer is the second leading cause of death worldwide and chemotherapy, radiotherapy, and/or surgery are the most common cancer treatment techniques.Over the past decade, much research has focused on finding new therapies that reduce the side effects of conventional treatments.
The identification of gene fusions in certain cancers has provided a practical target for expanding therapeutic options and advancing precision medicine.These genetic abnormalities lead to the expression of constitutively active fusion proteins that are carcinogenic drivers [1].Gene fusions are a type of mutation that commonly occurs in many types of cancer.They often result from chromosomal rearrangement that cause migration of coding or regulatory regions between genes.The tropomyosin tyrosine receptor kinase (TrK) family is of interest because the NTRK genes encoding have been implicated in gene fusions identified in a variety of adult and pediatric tumors.Three members of TrKA, encode transmembrane proteins NTRK1, TrKB (NTRK2) and TrKC (NTRK3) [2,3].As shown in Fig. 1, Trks are activated by the a family of nerve growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), Neurotrophin-4 (NT-4) and Neurotrophin-3 (NT-3) [3].
Larotrectinib is an inhibitor of the tropomyosin receptor kinases TrkA, TrkB, and TrkC (approved by the FDA in 2018).It has been indicated in adults and adolescents with solid tumors harboring NTRK gene fusions without a known acquired resistance mutations, in case of metastases or undergoing surgery.Resection can cause serious complications.Figure 2 shows another multitarget type-I kinase inhibitor with a pyrazole ring, such as entrectinib [4][5][6][7].Despite the high response rates achieved with firstgeneration TrK inhibitors, drug resistance still exists, ultimately leading to treatment failure [8,9].Additionally, TrKA is the most commonly identified oncogene, found in several tumor types at a rate of approximately 7.4% (4% for TRKB and 3.4% for TRKA) [10,11].
Furthermore, TrKA has been shown to mediate the stimulation of early tumor growth [12].Therefore, inhibiting TrKA signaling is an attractive clinical approach for cancer therapy.Therefore, it is highly desirable to obtain new selective Trk inhibitors with different chemical scaffolds as new anti-neuroblastoma (NB) agents.Previously, two TrKA inhibitors were approved by the U.S. Food and Drug Administration (FDA).Larotrectinib was approved for solid tumors with NTRK gene fusions in November 2018 [13], with very low IC 50 value for the Trk family (IC 50 = 2-20 nM), and significant activity outside this kinase family [14].Entrectinib was approved in August 2019 for NTRK gene fusion-positive or ROS1positive solid tumors [15].According to the classification of Shokat et al. [16] all are classified as type I kinase inhibitors.

Materials and methods
The melting points are uncorrected and measured on an Electrothermal instrument (9100).Infrared spectra were recorded on a Perkin Elmer 1430 spectrophotometer (KBr pellet).On a Varian Gemini NMR spectrometer using tetramethylsilane as the internal reference and the results are expected as δ value, the 1 H NMR and. 13C NMR spectra were recorded at deuterated dimethylsulfoxide at 300 and 75 MHz.Mass spectra were performed on a Shimadzu GCMS-QP 1000 Ex mass spectrometer at 70 eV.Elemental analysis was performed at the Center for Microanalyses of Cairo University, Giza, Egypt.Enzyme, cell cycle and apoptosis inhibition were performed at VACSERA, Cairo, Egypt.Compound 1 was prepared according to the previous literature [34]

General procedure of synthesis of 4a,b
In 15 ml of DMF, a mixture of compound 1 (0.01 mol) and diketones 2a,b (0.01 mol) containing few drops of piperidine was heated under reflux for 10 h.The resulting Fig. 3 Some pyrazolo [1,5-a]pyrimidines such as I-III with a standard drug dinaciclib as anticancer agents Fig. 4 Our target compounds as anticancer agents and TrKA inhibitors compared to larotrectinib solid was filtered, washed with ethanol and recrystallized from DMF. [4ʹ,3ʹ:3,4]pyrazolo [1,5-a]

General procedure of synthesis of compounds 9a-d and 11a-d
Method A: Compound 4a or 4b (0.01 mol) and sodium acetate (0.01 mol) were stirred in DMF (5 ml) under cooling in an ice-bath (0-5 °C).To the resulting cold solution is added portionwise a cold solution of the appropriate arenediazonium chlorides 8a-d.The mixture was stirred again under cooling conditions for 3 h., the resulting solid was filtered, washed with water and recrystallized from DMF.

General procedure for synthesis of compounds 14a,b
A mixture of compound 1 (0.01 mol) and β-ketoesters 12a,b (0.01 mol) was refluxed in glacial acetic acid (20 ml) for 9 h.The resulting solid was collected by filtration and recrystallized from DMF. [1,5-

General procedure for synthesis of compounds 15a-f
Refluxing of a mixture of compounds 14a,b (0.01 mol) and aldehydes 6a-c (0.01 mol) in DMF (20 ml) in a few drops of piperidine for 10 h.The solid that formed was filtered and crystallized from DMF.

General procedure of synthesis of compounds 16a-h
A cold solution of arenediazonium chlorides 8a-d was added drop wise in an ice-bath (0-5 °C) to a mixture of compound 4a (0.01 mol) and sodium acetate (0.01 mol) in DMF (5 ml), after stirring for 3 h.After forming, the resultant solid was filtrated, washed with water and recrystallized from DMF.   [1,5-a] [1,5-a]

Cell line
The three cell lines MCF7, HePG2 and HTC 116 were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt.Doxorubicin was used as a standard anticancer drug for comparison.

MTT assay
Determination of the inhibitory effects of compounds on cell growth was performed through the MTT assay [35,36].This colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells.The protocol was discussed in details in Additional file 1.

Tropomyosin receptor kinase A (TrKA) inhibitory assay
The TrkA assay Kit is designed to measure TrkA activity for screening and profiling applications using Kinase-Glo ® MAX as a detection reagent.The TrkA Assay Kit comes in a convenient 96-well format, with enough purified recombinant TrkA enzyme, TrkA substrate, ATP and kinase assay buffer for 100 enzyme reactions.The method was discussed in details in the ESI.

In-vitro cell cycle analysis
HepG-2 cells are pre-cultured in 25 cm 2 cell culture flask.RPMI-1640 medium was used.Tested compounds 7b, 9c, 15b, 16a and 16c were used in the cell treatment at their IC 50 by dissolving them in the required medium separately.The procedure was discussed in details in the ESI.

Annexin V-FITC apoptosis assay
HepG-2 cells were harvested and incubated with compounds 7b, 15b, 16a and 16c separately for 48 h.Then, the cells were collected and washed with PBS two successive times followed by centrifugation.After that, the cells were treated with Annexin V-FITC and propidium iodide (PI) using the apoptosis detection kit (BD Biosciences and Annexin V-FITC and PI binding were analyzed by a flow cytometer.

Molecular docking study
Molecular docking study was performed using program "Molecular Operating Environment (MOE) 2009.The protein structure was downloaded from the PDB data bank (http:// www.rcsb.org/ PDB codes: 5H3Q).The steps were discussed in details in the ESI.

Results and discussion Chemistry
Reactivity of 3-amino-1,7-dihydro-4H-pyrazolo[4,3-c] pyridine-4,6(5H)-dione 1 as a precursor of some heterocycles of interesting biological activity [24,39] encouraging us to continue our research on the synthesis of new compounds as a potential anticancer agents.Thus, condensation of compound 1 with each of acetylacetone 2a and dibenzoylmethane 2b, respectively, in N,N-dimethylformamide with a few drops of piperidine afforded products 4a, b.The structures of 4a, b were proven by spectroscopic techniques (Scheme 1).The IR spectrum of compound 4a shows absorption bands at 3187 and 1702 cm −1 assigned to the NH and CO groups, respectively.Its 1 H NMR spectrum revealed three singlet signals assigned to the two methyl and methylene protons at δ = 2.58, 2.70 and 4.05 ppm, respectively, in addition to a singlet signal at δ = 7.16 ppm for pyrimidine proton.In addition, the D 2 O exchangeable signal appeared at δ = 10.82 ppm corresponding to the NH proton.Furthermore, the mass spectrum of 4a displayed a molecular ion peak at m/z = 230 (M + , 67.6%), consistent with the molecular formula C 11 H 10 N 4 O 2 (Scheme 2).
The mechanism of the formation of compounds 4a, b was suggested to proceed through nucleophilic attack of the exocyclic amino group in compound 1 on the ketonic function of acetylacetone 2a, followed by intramolecular cyclization with elimination of water from the intermediate 3 to produce.The other pathway that leads to formation of compounds 5a, b was excluded as shown in Scheme 1.
Each of compounds 4a (and 4b) was condensed with the appropriate aromatic aldehyde 6a-j in refluxing DMF in presence of traces of piperidine to yield the respective arylmethylene derivatives 7a-t.The structures of 7a-t were supported by spectroscopic techniques.Compound 7a exhibits absorption bands in its IR chart at ν 3181 and 1698 cm −1 assigned to the NH and CO groups, respectively.Its 13 C NMR exhibited characteristic signals at 17.02 (CH 3 ), 128.34 (CH=C), 163.62 (CO) and 166.00 (CO), in addition to the expected signals (Scheme 2).
Further coupling of compound 4a with arenadiazonium salts 8a-d in DMF containing sodium acetate at 0-5 °C afforded the corresponding arylhydrazono derivatives 9a-d (Scheme 3).The resulting structures were established by elemental analysis and spectroscopic data.For example, the IR spectrum of 9b is characterized by the presence of absorption bands at 3187 and 1687 cm −1 due to the NH and CO groups, respectively.Also, in 1 H NMR spectrum appeared three singlet signals at δ = 2.30, 2.62 and 2.87 ppm due to three methyl groups, as well as two other singlet signals that can be exchanged with D 2 O at δ = 10.94 and 12.40 ppm due to two NH protons.The mass spectrum showed a molecular ion peak at m/z = 348 (M + , 100%), corresponding to the molecular formula C 18 H 16 N 6 O 2.. Compounds 9a-d were also obtained by an alternative chemical route by condensing aryl hydrazo derivatives 10a-d [24] with acetylacetone 3a under reflux conditions in DMF using piperidine as basic medium (Scheme 3).
Similarly, 4b coupled with arenadiazonium salts 8a-d under the same reaction conditions to produce arylhydrazono derivatives 11a-d (Scheme 4).The structures generated are supported by spectroscopic data (see exp. (Scheme 5), which were confirmed by spectroscopic tools.The IR spectrum of compound 14a was characterized by the presence of absorption bands at 3430, 3187 and 1689 cm −1 assigned to the OH, NH and CO groups, respectively.The 1 H NMR spectrum also revealed a singlet signals assigned to methyl, methylene, pyrimidine-H, NH and OH protons at δ = 2.37, 3.95, 5.86, 10.96, and 12.80 ppm, respectively.The mass spectrum also exhibited a molecular ion peak at m/z = 232 = (M + , 100%), confirming that the molecular formula C 10 H 8 N 4 O 3. The mechanism of formation of 14 is thought to occur initially by nucleophilic attack of the exocyclic amino group on 1 into the ketonic function of β-ketoesters 12a,b leading to the elimination of water molecule, followed by the intramolecular cyclization, followed by elimination of the ethanol molecule to obtain the enol structure 14 instead of the keto form 13 as shown in Scheme 5.
Condensation of each of 14a,b with the suitable aromatic aldehyde 6a-c in DMF under reflux conditions using a few drops of piperidine yielded the respective arylmethylene derivatives 15a-f (Scheme 6).The IR spectrum of 15a presented absorption bands at 3419, 3203 and 1704 cm −1 assigned to OH, NH and CO groups, respectively.Its 13   The coupling reaction of compounds 14a,b with arenediazonium chlorides 8a-d in DMF containing sodium acetate at 0-5 °C yielded the corresponding arylhydrazono derivatives 16a-h.The structure of 16a-h was determined by elemental analysis and spectral data.The IR spectrum of compound 16c was characterized by the presence of absorption bands at 3426, 3195 and 1702 cm −1 owing to the OH, NH and CO groups, respectively.Moreover, 1 H NMR chart of compound 16c appeared two singlets at δ = 2.41 and 3.76 ppm due to methyl and methoxy protons along with three other singlet signals exchangeable with D 2 O in the region 11.19, 12.37 and 12.87 ppm due to three protons of 2NH and OH.The mass spectrum also showed a molecular ion peak at m/z = 366 (M + , 49.7%), which confirmed its molecular formula C 17 H 14 N 6 O 4 (Scheme 7).

Anticancer activity
Compounds 1, 4a,b, 7a-c, 7k, l, 9a-c, 14a, 15b, 16a-c were selected to be investigated against three human cancer cell lines MCF7, HepG2 and HCT116 cell lines using MTT assay using doxorubicin as the standard drug.Each point is the mean ± SD (standard deviation) of three independent experiments performed in triplicate, using the prism software program (integrated Graphpad software, version 3).Cytotoxicity was assessed at concentrations of 5, 10 and 20 µg/l and the IC 50 values of the tested compounds compared to the reference drug were evaluated as shown in Table 1, 2 and 3.In addition, the percentage of the viable cells was measured and compared with the control (Figs.5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20).From the results presented in Table 1, compounds 7b and 16c strongest cytotoxic activity against MCF7 with IC 50 = 3.864 and 3.805 µg/l, respectively, among the tested compounds compared to the doxorubicin (IC 50 = 2.527 µg/l).Other compounds tested showed moderate to weak cytotoxic activity.
Furthermore, from screening the cytotoxic activity of the tested compounds against HepG2 cell line, we can infer that, compounds 7b, 15b, 16a and 16c showed higher potency against the HepG-2 cell line with IC 50 = 4.250, 4.641, 3.555 and 3.427 µg/l, respectively compared to the reference drug (IC 50 = 4.749 µg/l).The remaining tested compounds showed moderate to weak activity (Table 2).
Based on the results of the cytotoxic activity of the tested compounds against HCT116 (Table 3), compound 7b exhibited a higher cytotoxic activity against Scheme 7 Synthesis of arylhydrazono derivatives 16a-h    The results obtained from the anticancer activity of some newly prepared compounds show the all tested compounds have antitumor activity against all the three cell lines (MCF7, HepG2 and HCT116) (Fig. 21).Initially, parent compound 1 exhibited a moderate cytotoxicity (IC 50 = 7.818 µg/l) against the HepG2 cell line compared to doxorubicin (IC 50 = 4.749 µg/l).
When compound 1 was converted into a tricyclic ring system containing a pyrimidine ring as in compounds 4a,b, the anticancer activity varies depending on the nature of the substituents present on the pyrimidine ring.Therefore, when the substituent of compound 4 is a methyl group like 4a, the anticancer activity gradually increases towards HCT116 with an IC 50 of 3.966 µg/l, equivalent to doxorubicin (IC 50 = 3.641 µg/l).When adding another aryl group to 2nd position of compounds 4a,b, the anticancer activity changes depending on the position of the substituents on the aryl group.Thus, the anticancer activity does not change when the aryl group is a phenyl ring.However, when the methoxy group was introduced as a donor group in the aryl moiety as in 7b, the anticancer activity increased towards MCF7 (IC 50 = 3.864 µg/l), HepG2 (IC 50 = 4.250 µg/l) and HCT116 (IC 50 = 2.487 µg/l) as shown in Tables 1, 2 and 3. On the other hand, when compounds 7a,b contain a chlorine atom on the aryl group as in the case of 7c, the anticancer activity was reduced in all three cell lines tested.Coupling of compounds 4a,b with arenediazonium salts afforded arylhydrazo derivatives 9a-d which, have different cytotoxicities depending on the nature of the substituent on the arylhydrazo moiety.Thus, when the arylhydrazo was a phenyl or tolyl group, it had low anticancer activity against all three cell lines, whereas for the aryl moieties containing a methoxy group as donating group like 9c, the anticancer activity increased especially in the HCT116 cell line with IC 50 = 3.778 µg/l.Furthermore, when compound 1 was condensed with a β-ketoester to form a tricyclic ring system containing a hydroxyl group as in 14a,b, it appeared to have weak anticancer activity.However, when compounds 14a,b have a methoxy group as in 15b, the anticancer activity was increased against HepG2 with IC 50 = 4.641 µg/l compared to doxorubicin (IC 50 = 4.749 µg/l).Also, when compounds 14a,b were coupled with arenediazonium salts to afford arylhydrazo derivatives 16a-c, the anticancer activity was increased in the case of the arylhydrazo group with the   1, 2, and 3).

Enzyme inhibition assay
Compounds 7b, 9c, 15b, 16a and 16c with the strongest anticancer activity were tested for tropomyosin kinase A receptor inhibitory activity by a kinase assay technique utilizing Larotrectinib as a positive control.The data listed in Table 4 and Fig. 22 demonstrate that compound 16c has the strongest inhibitory effect among the tested compounds against to tropomyosin receptor kinase A (TrKA) with IC 50 = 0.047 ± 0.0027 μg/ml compared to Larotrectinib with IC 50 = 0.034 ± 0.0021 μg/ ml using the HepG2 cancer cell line.While, compounds 7b and 16a have moderate activity anti-TrKA with IC 50 = 0.064 ± 0.0037 and 0.072 ± 0.0042 μg/ml, respectively.In addition, compounds 9c and 15b have weak activity against TrKA with IC 50 = 0.158 ± 0.0092 and 0.101 ± 0.0059 μg/ml, respectively.Therefore, compound 16c can cause cancer cell line death by inhibiting the enzyme tropomyosin receptor kinase A, possibly because it contains a methoxy group as donating group.

Cell cycle analysis
For cell cycle analysis, stained DNA from HepG2 cancer cells was treated with compounds 7b, 15b, 16a and 16c  5, it can be seen that the proportion of cells at phase in the pre-G1 of compounds 7b, 15b, 16a and 16c increased the proportion of cells at phase in the G2/M by about 4, 4, 2.5 and 3 folds, respectively.

Detection of apoptosis assay
Early and late apoptosis was determined after treatment of HepG2 cells with compounds 7b, 15b, 16a and 16c compared with untreated control cells.The late apoptosis rate increased by about 13, 20, 4 and 3 times, respectively, showing a higher efficiency than the early apoptosis ratio 5, 2, 8 and 6 times, respectively.Total apoptosis from treatment of HepG2 cells with compound 15b showed the higher apoptotic induction efficiency compared with other tested compounds 7b, 16a and 16c (Table 6 and Fig. 29).

Molecular docking study
The most potent inhibitory compounds 7b, 16a and 16c as well as the standard drug Larotrectinib against TrKA were docked with the crystal structure of tropomyosin receptor kinase A (TrKA) (PDB: 5H3Q, Fig. 30) used the molecular operating environment docking (MOE)  2009 to find the exact binding pattern to the receptor.From the present studies, it was found that all the anchored compounds exhibited good binding energies ranging from − 7.3801 to − 6.5837 kcal mol −1 and displayed good fitness with the active site of the 5H3Q protein.Thus, the standard drug Larotrectinib exhibits two hydrogen bond interactions with bond length 2.99Ǻ and 3.06 Ǻ with amino acid residues Lys 544 and Asp 668, respectively and binding energy (S) = − 7.1325 kcal mol −1 (Fig. 31).Compound 7b appears to have a hydrogen bond interaction with a bond length 2.98 Ǻ between the carbonyl function of the pyridine moiety and the amino acid residue Lys 544 as well as a cation-cation interaction between the 4-methoxyphenyl group and His 489 with S = − 7.3801 kcal mol −1 (Fig. 32).On the other hand, compound 16a exhibits a binding energy of S = − 7.0296 kcal mol −1 and appears two hydrogen bond interactions with bond length equal to 3.12 and 3.46 Ǻ between the two carbonyl groups of each of pyridine and pyrimidine rings, respectively and the amino acid residues Lys 544 and Arg 673 (Fig. 33).Additionally, compound 16c the most potent inhibitory activity against TrKA exhibits two hydrogen bond interactions, one between the carbonyl group of the pyrindine ring and Met 507 with bond length of 3.52 Ǻ and the other between the carbonyl group of the pyrimidine ring and Asp 596 with bond length equal to 3.17 Ǻ, as well as a cation-cation bond interaction between pyrazole ring and Val 524 with S = − 7.4667 kcal mol −1 (Fig. 34).All data presented from the molecular docking study for larotrectinib, 7b, 16a, and 16c are listed in Table 7.

In silico ADME studies
In silico prediction of potential pharmacokinetic properties absorption, distribution, metabolism and excretion toxicity (ADME/T) properties calculated using Swiss ADME (http:// www.swiss adme.ch/) online tools are presented in Table 8.Some physical properties such as absorption, distribution, metabolism, excretion and    rule, the compounds 4a, 7a-c, 9c, 15b, 16a, 16b and 16c have violated all parameters of Lipinski's rule of five.The results listed in Table 9 show that all the compounds have TPSA values and compounds 4a, and 7a-c have optimal topological polar surface area (TPSA) of 76.36, 76.36, 85.59, and 76.36 Ǻ 2 , respectively.This means that compounds 4a, 7a, 7b and 7c are better able of permeate cell membranes and adhere to RO5 and are well absorbed through the gastrointestinal tract.In silico predictions of toxicological properties were determined using the Osiris property explorer program (http:// www.prper ty explorer-cheminfo.org)online tools is presented in Table 9.In the toxicological profile of the compounds, 9c and 16c may exhibit medium tumorigenicity, but compounds 7b, 9c, 15b, 16a and 16c are high risk in the reproductive system is expected.Additionally, all compounds have no irritant effects.Compounds 4a, 7a, and 7c did not cause the toxicity problems mentioned in the software used in this study.All of the compounds studied have positive drug-likeness values, meaning that they all contained fragments commonly found in commercial drugs (Table 9).Swissadme helps us provide information on poorly and highly absorbed drugs to model passive intestinal absorption through the human intestinal tract.Graphical prediction of intestinal absorption and blood-brain barrier permeation of the most potent anticancer activity compounds 4a, 7a, 7b, 7c, 9c, 15b, 16a, 16b and 16c against MCF7, HepG2 and HCT116 are shown in Fig. 35.Boiled egg diagram showing the bioavailability property space for wlog P and TPSA [white area means that intestinal absorption; The yellow area means it has entered the brain well, the intestinal are well absorbed; and the gray area means the intestinal have poor absorption].This provides a simple visual cue to profile new compounds for their oral absorption.All compounds studied were found in the white region.Additionally, PGP + (substrate) and PGP − (nonsubstrate) are denoted by blue and red dots for molecules predicated to be CNS efflux or not efflux by P-glycoprotein, respectively.Therefore, all the studied compounds 4a, 7a, 7b, 7c, 9c, 15b, 16a, 16b and 16c are not substrates of P-gp (PGP-), hence, we can say that these compounds have good bioavailability.In this series, compound 7c gave BBB and a low TPSA of 76.36.This suggests that the molecule can be absorbed very easily through the gastrointestinal tract and preferentially acts as a hydrophobic agent and can be easily transported across the blood-brain barrier.

Conclusion
In this study, a novel series of pyrido[4ʹ,3ʹ:3,4] pyrazolo [1,5-a]pyrimidine derivatives were synthesized.The anticancer activity of these compounds was tested on MCF7, HepG2 and HCT116 cell lines in comparison to doxorubicin.The results showed that some of the synthesized compounds have significant cytotoxic activity.Compound 7b exhibited high and broad spectrum anticancer activity against all cell lines tested.TrKA inhibition assays on 7b, 9c, 15b, 16a and 16c showed a decrease in TrKA expression with IC 50 values below 0.2 μg/ml.The most potent anticancer targets were examined for their effects on cell cycle distribution and apoptosis induction.The results revealed that 7b and 15b induced arrest at the G2/M phase of the cell cycle

Compd. no Mutag Tum
in HepG2 cells among the other tested compounds.Furthermore, docking studies revealed that 7b, 16a and 16c bind with high affinity to the active site of TrKA.In addition, compounds 7b, 15b, 16a and 16c appear to be well absorbed from the gastrointestinal tract.These results suggest that these compounds may be a promising tools for the production of more potent anticancer agents.

Fig. 30 Fig. 31 Fig. 32
Fig. 30 Interaction of 5H3Q with the active site in 2D and 3D

Table 1
The IC 50 values (the drug concentrations that inhibited 50% of cell proliferation) of the compounds on MCF7 cell line the HCT116 (IC 50 = 2.487 µg/l) compared to the doxorubicin (IC 50 = 3.641 µg/l).Additionally, compounds 7c, 16a and 16c exhibited high anticancer activity against HCT116 with IC 50 values of 4.072, 4.369 and 4.503 µg/l, respectively.The other rested compounds showed moderate to low activity.

Table 2
The IC 50 values (the drug concentrations that inhibited 50% of cell proliferation) of the compounds on HepG2 cell line

Table 3
The IC 50 values (the drug concentrations that inhibited 50% of cell proliferation) of the compounds on HCT-116 cell line

Table 6 The
effect of compounds 7b, 15b, 16a and 16c on HepG2 cell lines

Table 8
Important pharmacokinetic parameters for bioavailability of compounds 4a,

Table 9
Important toxicity predication of compounds 4a,